Process for preparing a sterile high molecular weight hyaluronic acid formulation

ABSTRACT

A process for preparing a sterile ready-to-use aqueous pharmaceutical formulation comprises a high molecular weight hyaluronic acid salt (HA) at a specified concentration, comprising the steps of: providing an aqueous formulation comprising high molecular weight HA at a concentration of less than the specified final concentration; passing said aqueous formulation through a filter having a pore sizeless than 0.45 pm; concentrating said aqueous formulation by applying a vacuum and boiling off water until said specified concentration is reached.

Applicant claims foreign priority benefits under 35 U.S.C. §§119(a)-(d)or (f), or §365(b) of European Patent Application No. 02405681.4, filedAug. 7, 2002.

The present invention relates to a process for preparing a sterile highmolecular weight hyaluronic acid salt as a final formulation forpharmaceutical use.

Hyaluronic acid in its salt form may for instance include sodiumhyaluronate, potassium hyaluronate, magnesium hyaluronate, calciumhyaluronate, or others.

Hyaluronic acid is a mucoid polysaccharide of biological origin, whichis widely distributed in nature. For example, it is known thathyaluronic acid is present in various animal tissues such as umbilicalcord, synovial fluid, vitreous humor, rooster comb and variousconnective tissues such as skin and cartilage.

Chemically, hyaluronic acid is a member of glycosaminoglycans and it isconstituted by alternating and repeating units of D-glucuronic acid andN-acetyl-D-glucosamine, to form a linear chain having a molecular weightup to 13×10⁶ Daltons.

In the meaning of the present invention, high molecular weighthyaluronic acid is hyaluronic acid having an average molecular weight ofnot less than 0.5×10⁶ Daltons.

Pharmaceutical use of hyaluronic acid or of a salt thereof is widelydescribed in the literature.

Since hyaluronic acid is a non-immunogenic substance and hasviscoelastic and hydrophilic properties, it is used, since severalyears, as an eye vitreous or joint fluid replacement or as a supportivemedium in ophthalmic surgery, as disclosed for example in U.S. Pat. No.4,141,973 of Balazs.

In joint fluids, the viscous hyaluronic acid solution serves as alubricant to provide a protective environment to the cells, and for thisreason, it is used in the treatment of inflamed knee joints.

EP-A-0 781 547 discloses a sodium hyaluronate based ophthalmicformulation for use in eye surgery.

EP-A-0 719 559 discloses sodium hyaluronate viscous solutions for use asmasking fluid in therapeutic photokeratectomy by means of excimer laser.

EP-A-0 875 248 discloses the use of hyaluronic acid or of one of itspharmaceutically acceptable salts for the preparation of an aqueoussolution useful as intra-articular lavage liquid.

EP-A-0 698 388 of Chemedica S. A. discloses an ophthalmic preparationfor use as artificial tears containing hyaluronate as a viscositythickener.

The pharmaceutical use of hyaluronic acid or of a salt thereof requiresa highly pure and sterile product.

Hyaluronic acid can be extracted and purified from animal or microbialsources such as umbilical cords, rooster combs or from group A and CStreptococci as disclosed for example in U.S. Pat. No. 4,141,973 ofBalazs, U.S. Pat. No. 5,559,104 of Romeo et al. and WO 00/4925.

Industrial extraction and purification processus of hyaluronic acidtypically produce hyaluronic acid salts, such as sodium hyaluronate, inthe form of a dried powder. The purified pharmaceutical grade driedpower may be used for preparing, for example, aqueous pharmaceuticalformulations for the various pharmaceutical uses such as interarticularinjection, eye drops or vitreous humor replacement.

A common industrial process for preparing ready-to-use pharmaceuticalformulations comprises the mixing of a defined quantity in weight ofsodium hyaluronate with a precise volume of water and, as the case maybe, salt such as sodium chloride and buffers such as phosphates andother excipients. As the concentration and composition of theformulation for pharmaceutical use should remain within a narrowlydefined range, the various components of the formulation are carefullymeasured. The formulation is then filled into recipients such assyringes and vials of defined dosages ready for use. Subsequent tofilling of the recipients, the formulation is sterilized by autoclavetypically at around 121° C. for fifteen minutes or more.

One of the problems with the use of heat to sterilize hyaluronic acid isthe known effect on breaking the molecular chains forming HA, thusreducing the average molecular weight of HA.

The high molecular weight of hyaluronic acid is an importantpharmacological property.

In many pharmaceutical applications it is undesirable to have lowmolecular weight hyaluronic acid in the formulation, for example in viewof the inflammatory effects of low molecular weight HA as reported inU.S. Pat. No. 4,141,973 and the loss of beneficial reological propertiesof high molecular weight HA. In order to compensate for degradation ofthe HA in a formulation of given concentration in the aforementionedsterilization methods, the hyaluronic acid or salts thereof initiallyused in preparing the formulation have an average molecular weight thatis higher than that of the desired minimum average molecular weight ofthe final formulation. This is however uneconomical since the yield ofhyaluronic acid from starting material decreases as the averagemolecular weight required increases.

Another known method of sterilizing hyaluronic acid is by filtration.This technique is used in conventional industrial processes forpreparing purified hyaluronic acid salts in a concentrated form, usuallyin the form of dried powder, whereby a low concentration aqueoussolution is passed through the filter and subsequently dried.

Such sterilization steps are for example described in European patentapplication EP 867453 and in PCT application WO 00/44925. In theseapplications, a filter with a pore size as small as 0.22 μm is alsodisclosed for sterilization. Filters having a pore size of 0.22 μm havea bacterial challenge of 1 over 10⁷ bacteria, based on the smallestknown bacterium Pseudomonas diminuta, while filters having a pore sizeof 0.45 μm have a bacterial challenge of 1 over 10⁴ bacteria (alwaysbased on Pseudomonas diminuta). For this reason, filters having a poresize less than 0.45 μm are considered to be sterilizing.

In conventional industrial processus, the method of sterilization byfiltration is not known to be used for preparing high viscositypharmaceutical formulations ready for use, since at the requiredconcentration of HA in high viscosity pharmaceutical aqueousformulations, typically in the range of 1 to 2% wt/v, not all of thehyaluronic acid passes through the filter at 0.22 μm. Since this resultsin a change in the concentration and/or a loss of hyaluronic acid,sterilization by filtration for preparing ready-to-use high viscositypharmaceutical formulations is problematic.

In U.S. Pat. No. 5,093,487, the preparation of an aqueous pharmaceuticalformulation comprising high molecular weight sodium hyaluronate readyfor use and sterilized through a filter of 0.22 μm is described. Thesterilizing method described in this patent however relies on a numberof passes of hyaluronic acid aqueous formulation through the 0.22 μmfilter, so as to irreversibly reduce the viscosity of the hyaluronicacid. The sterilization of an aqueous pharmaceutical formulationcomprising HA at a concentration of 1% or more is, according to thispublication, possible in view of the reduction of viscosity of thehyaluronic acid resulting from the multiple passes through the filter.It is further argued in this application that the viscosity is reducedwithout reducing the molecular weight of HA. Without wishing to takeposition on the validity of the findings reported in the aforementionedpublication, for many pharmaceutical applications such asintra-articular applications, the lowering of the viscosity of HA isundesirable.

An object of the present invention is to obtain a sterile ready-to-usepharmaceutical aqueous formulation comprising a hyaluronic acid salt,that is sterile and economical to produce, particularly in industrialconditions. It is advantageous to provide such formulation with a narrowtolerance in the concentration of the ingredients of the formulation.

Objects of this invention have been achieved by a process for preparinga sterile high molecular weight hyaluronic acid formulation forpharmaceutical use according to claim 1.

Disclosed herein is a process for preparing a sterile ready-to-useaqueous pharmaceutical formulation comprising a high molecular weighthyaluronic acid salt (HA) at a specified final concentration, comprisingthe steps of:

-   -   providing an aqueous formulation comprising high molecular        weight HA at a concentration less than the specified final        concentration;    -   passing said aqueous formulation through a filter having a pore        size less than 0.45 μm;    -   concentrating said aqueous formulation by applying a vacuum and        boiling off water until said specified final concentration is        reached.

Advantageously, the reduced concentration of the aqueous formulationprior to filtering, as a function of the molecular weight, reduces theviscosity and enables the entire HA to pass through the filter and besterilized, the subsequent boiling with a vacuum ensuring thatessentially no heat dependent degradation of HA occurs. The process iswell adapted to an industrial environment and is particularlyeconomical, allowing ready-to-use dosages of pharmaceutical formulationcomprising HA to be filled in sterile recipients without furthersterilization.

A further advantage is that the formulation is microbiologically stableand may be kept for weeks before being used as pharmaceuticalpreparation.

Viscosity of HA in aqueous solution is a property that depends onseveral parameters such as molecular weight, concentration, temperature,concentration and quality of salts, pH and shear rate applied to thesolution. Higher molecular weight and concentration increase theviscosity, while higher shear rate and salts decrease the viscosity.Regarding the temperature, HA has a hysteretic behavior as reported byM. Cardones et al. “Hysteresis Behavior of Sodium Hyaluronate Solutionsduring Heating and Cooling”, Clear Solutions Biotech, Inc., TechnicalRep 01. Viscosity increases and decreases in an irregular way byincreasing or decreasing the temperature. Between 60° C. and 70° C. itis possible to get the minimum viscosity of the solution. Therefore,this hysteretic property can be used for decreasing or increasing theviscosity of the solution, but it has to be considered that the HAchains degradation is proportional to the temperature and the durationat which this temperature is kept.

During the concentrating step after filtration, the concentration of HAmay be monitored in real time in order to stop the vacuum boiling whenthe specified concentration for the ready-to-use pharmaceuticalformulation is reached. The monitoring or measuring process mayadvantageously be carried out with a spectrophotometer with the sensingbeam placed in the formulation, the absorption of radiation in theultraviolet range (UV) being proportional to the HA concentration. Aparticularly advantageous feature of this invention is that it obviatesthe need to mix exact quantities of water and hyaluronic acid to obtainthe specified concentration and ensure that such concentration ismaintained through the process. Instead, the initial HA and water mixhas an approximate concentration lower than the final formulation, thussimplifying the process.

The vacuum applied during the concentrating process is preferably lessthan 200 millibars absolute pressure, in particular in the range of 30to 60 millibars, for example 40 millibars, whereby the boilingtemperature is around 26 to 280° C. Industrial equipment is economicalto operate reliably at such pressures, and the low temperature avoidsany significant or measurable reduction of the hyaluronic acid molecularweight.

The filter pore size is advantageously around 0.22 μm or less, thusensuring the preparation of a highly sterile formulation. Filtersterilized ready-to-use high viscosity pharmaceutical formulation withHA concentration in the range of 1 to 3% can thus advantageously beprepared in a sterile and economical manner, according to thisinvention.

Further advantageous aspects of this invention will be apparent from theclaims and the following detailed description of an example of a processand annexed drawing in which:

FIG. 1 represents a flow chart illustrating steps in an embodiment ofthe process according to the invention;

FIGS. 2 and 3 are graphs represent the percentage of HA of molecularweight 1.1 million and 2.2 million Daltons, respectively, passingthrough a filter of 0.22 μm as a function of the % w/v concentration inan aqueous solution, at 20° C. and neutral pH;

FIG. 4 is a graph representing the optical density (OD) at 230 nm of theformulation as a function of time during the concentration process asmeasured by a spectrophotometer.

Referring to FIG. 1, a process for preparing a ready-to-use aqueouspharmaceutical formulation comprising a high molecular weight hyaluronicacid salt at a specified pharmaceutical concentration is shown. Prior topreparing the formulation, the process plant equipment is cleaned withpurified water (step 1), sterilized with clean steam (step 2), andwashed with sterile distilled water for injection (WFI) (step 3).

A preparation of an aqueous HA formulation, for example a diluted sodiumhyaluronate solution at a concentration less than the concentrationspecified for the final pharmaceutical formulation is introduced into amixing reactor for preparing the prefiltered formulation (step 4).

The aqueous HA solution may either be prepared from a dried HA salt orfrom the solution prepared according to PCT application WO 00/44925,before the drying process.

The addition of a concentrated salt solution (25×), dosed by aperistaltic pump, is carried out in order to add the right amount ofsalts coupled with the amount of HA added (step 5). The salt solutionnormally contains NaCl, buffers and other excipients specified for thefinal pharmaceutical formulation, and they are added in order to bringthe pH to a physiological pH, such as 7.4, and to give the finalformulation the physiological osmolarity, such as 300 mOsm/lt.

The salt solution, buffers and other excipients may also be addedaseptically after the filtration step 7 directly into the reactionchamber. This is particularly advantageous for very high molecularweight HA in view of the fact that salts decrease the filterability ofHA and thus would require greater dilution of the pre-filtered solution.The amount of excipients added can be monitored in real time by anelectrical conductivity sensor (probe) in the reaction chamber. Theconductivity of the HA formulation is related to the amount of excipientin the formulation and can be determined empirically. Thus, by measuringthe conductivity of the HA formulation as excipient is added in thereaction chamber until the required concentration is reached, therequired amount of excipient in the formulation can be added in asimple, reliable and precise manner. The aforegoing in particularobviates the need to calculate and measure dosages in advance, and inparticular removes the problem of having to take into account andcompensate for the certain amount of HA, even if small, retained by thefilter during the filtration step 7.

By way of example, the formulation may be prepared from 45 grams ofdried sodium hyaluronate of an average molecular weight of 2.2 millionDaltons mixed with 15 liters of WFI. In this case, due to the highmolecular weight of HA, the salts and buffer solution is added afterfiltration in order to avoid a greater dilution of the pre-filteredsolution than needed, as mentioned above.

A stirring machine in the reactor mixes the prefiltered solution (step6), for example for about 120 minutes, until it is homogeneous. In thisparticular example, the prefiltered solution has an HA concentration of0.3% wt/v. At this concentration, hyaluronic acid with a molecularweight of 2.2 million Daltons, at room temperature and physiological pH,has a low viscosity which enables it to pass entirely through a filterwith pore size of 0.22 μm. The maximum viscosity at which all of the HAformulation passes through a filter having a pore size of 0.22 μm, isfound to be approximately 5 Pa·s as measured at 0.1 s⁻¹ shear rate at20° C. This maximum viscosity however will depend on the filter poresize: a smaller pore size, such as 0.1 μm, would lower this maximumviscosity at which all HA would pass through the filter.

As discussed above, the maximum concentration of HA in an aqueoussolution at which substantially all the HA will pass through asterilizing filter, for example a filter of 0.22 μm, will depend on themolecular weight of the hyaluronic acid. This may be shown withreference to the graphs shown in FIGS. 2 and 3, whereby FIG. 2 shows thepercentage of sodium hyaluronate at average molecular weight of about1.1 million Daltons passing through a filter of 0.22 μm pore size atsubstantially ambient temperature (around 20° C.) and substantiallyneutral pH (around 7) as a function of the concentration (grams ofsodium hyaluronate per deciliter of water % wt/v).

FIG. 3 shows a similar graph under the same conditions except that thesodium hyaluronate has an average molecular weight of about 2.2 millionDaltons. It may be seen on the graph of FIG. 2 that all the sodiumhyaluronate at a molecular weight of 1.1 million Daltons passes throughthe filter up to a concentration of about 0.92% wt/v (or g/dl), whereasfor sodium hyaluronate having a molecular weight of 2:2 million Daltons,the maximum concentration is approximately 0.32% wt/v. Therefore, as themolecular weight of the hyaluronic acid salt increases from 1.1 to 2.2million Daltons, the maximum concentration of HA in an aqueous solutionto pass 100% through the 0.22 μm filter will decrease from about 0.94%wt/v to about 0.32% wt/v.

In an industrial process, and considering that after filtration thesolution is concentrated, it is preferred to have a concentrationsomewhat lower than the upper limit to ensure that the filtering processis complete and reliable with a certain margin for error or variationsfrom batch to batch in the molecular weight of the sodium hyaluronate,the temperature, and the mixing concentrations.

The solution may be forced through the sterilizing filter (step 7) byintroducing a gas under pressure, such a nitrogen, for example at around3 bars pressure in the mixing reactor, or by means of a pump. Thesterilizing filter preferably has a pore size of 0.22 μm, but filtershaving other pore sizes less than 0.45 μm and greater than around 0.1μm, may also be used to the extent such filters are or becomecommercially available.

When all the solution has passed through the filter into a distiller,the distiller is sealed off from the filter and mixing reactor with avalve, and a vacuum pump is activated and regulates the pressure in thedistiller by means of a regulatory valve (steps 8, 9, 10). The pressureis less than 200 millibars in order to bring the boiling temperaturebelow 60° C., but preferably the pressure is in the region of 30 to 60millibars, for example at 40 millibars, whereby the boiling temperatureof water is in the region of 26 to 28° C., close to ambient temperature.

Instead of the distiller, a thin film evaporator or any otherunder-vacuum concentrators can be useful for a batch, fed-batch orcontinuous concentration of the HA formulation.

A heating jacket around the distiller supplies heat energy during theboiling process. Advantageously, the boiling temperature of less than30° C. ensures that there is essentially no degradation of thehyaluronic acid such that the molecular weight of the hyaluronic acid isnot reduced.

An HA concentration sensor, advantageously in the form of aspectrophotometer having an optical fibre immersed in the formulation,may be provided to measure in real time the concentration of hyaluronicacid (step 11) and automatically stops the boiling process when thespecified concentration is reached. The spectrophotometer may forexample be based on the absorption of a beam of ultraviolet light (e.g.wave length 230 nm) positioned within the solution in the distiller.FIG. 4 shows the optical density at 230 nm measured over time during thewhole concentration process (for example up to 2% w/v.) The real time HAconcentration measurement obviates the need to mix very precisequantities during the preparation of the diluted prefiltered solution,thus simplifying the process.

The vacuum boiling also has an important advantage of degassing theformulation which, in view of the mixing process and application ofnitrogen at high pressure during the filtering process, comprisesbubbles, microbubbles and dissolved gas that are not acceptable in thefinal pharmaceutical formulation.

When the HA concentration sensor in the distiller signals that thespecified concentration is attained, the pressure in the distiller israpidly increased by introducing gas, for example nitrogen, therein(step 13) and the jacket heating around the distiller is cooled toambient temperature or less, thus immediately stopping the concentrationprocess of the formulation (step 12).

It may be noted that with a process according to this invention, theconcentration of hyaluronic acid in the formulation may be in the rangeup to 3%, depending on the specified pharmaceutical use.

The formulation can then be pumped or pushed by a gas (e.g. nitrogen)under pressure into sterile tanks (steps 28, 30) for filling at anotherlocation and/or in a subsequent stage directly into sterile recipients,such as syringes (step 16), ready for use. The pharmaceuticalformulation may also be directly filled into sterile recipients forpharmaceutical use without intermediate storage in a sterile tank.

1. A process for preparing a sterile ready-to-use aqueous pharmaceuticalformulation comprising a high molecular weight hyaluronic acid salt (HA)at a specified concentration, comprising the steps of: providing anaqueous formulation comprising high molecular weight HA at aconcentration of less than the specified concentration; passing saidaqueous formulation through a filter having a pore size less than 0.45μm; and greater than 0.1 μm; concentrating said aqueous formulation byapplying a vacuum and boiling off water until said specifiedconcentration is reached; and after the concentration step, filling thepharmaceutical formulation directly into sterile recipients ready forpharmaceutical use, or into sterile tanks and subsequently directly intosterile recipients ready for pharmaceutical use.
 2. Process according toclaim 1, wherein the vacuum is at a pressure in the range of 30 to 60millibars.
 3. Process according to claim 1, wherein the averagemolecular weight of HA is in the range of 800,000 to 5,000,000 Daltons.4. Process according to claim 1, wherein the filter has a pore size inthe range of 0.22 μm to 0.1 μm.
 5. Process according to claim 1,wherein, during the concentration step, the concentration of HA ismeasured in real time and the vacuum boiling process is stoppedautomatically when the specified concentration is measured.
 6. Processaccording to claim 1, wherein the HA concentration is measured with aspectrophotometer sensing wave radiation absorption in the formulation.7. Process according to claim 1, wherein excipients are added to theformulation after the filtration step, and wherein the conductivity ofthe HA formulation is measured in real time until the amount ofexcipients reaches a required value.